Plant fermentation composition for enhancing scalp immune barrier and melanocyte activity as well as preparation method and application of plant fermentation composition

Abstract

The invention discloses a plant fermentation composition for enhancing scalp immune barrier and melanocyte activity and a preparation method and application of the plant fermentation composition, and belongs to the technical field of microbial fermentation and skin immunization. Performing fermentation by utilizing Wickerhamomyces anomalus and saccharomyces cerevisiae; the mass ratio of the Wickerhamomyces anomalus to the saccharomyces cerevisiae is (3-10): 1; the weight ratio of the radix polygoni multiflori to the radix asparagi to the black tea is (10-30): (1-10): 1. The invention provides a cosmetic raw material for promoting melanogenesis by enhancing innate immune response of melanocytes by utilizing a microbial fermentation technology, and solves the technical problem that black hair becomes white. The cosmetic provided by the invention can improve the health of hair and scalp, has a good hair blackening effect, and effectively prevents the hair from becoming grey and white.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation and skin immunity, and specifically relates to a preparation method and application of a plant fermentation composition for enhancing scalp immune barrier and melanocyte activity. Background Art

[0002] Graying of hair is a common hair disease in the middle-aged and elderly population. It is caused by factors such as aging-induced immune decline, decreased melanocyte activity, and decreased tyrosinase activity, which lead to the obstruction of melanin synthesis. At present, there is no specific treatment for this disease in clinical practice, and most people use hair dye to cover it up. However, hair dyeing has a great impact on physical health. Hair dyes cannot fundamentally change the hair quality and may induce various diseases, such as contact dermatitis, leukemia, osteoporosis, and even cancer. Therefore, developing a safe and effective treatment method is crucial to solving the problem of graying in the middle-aged and elderly population.

[0003] Studies have found that melanocytes have a dual role in immune defense and melanin synthesis. Melanocytes, like other epidermal resident cells, play a role in enhancing the innate immune inflammatory response: melanocytes perceive environmental stimuli by expressing Toll-like receptors and participate in the innate immune response. Subsequently, the activation of Toll-like receptors (such as TLR4) can enhance melanin synthesis. In addition, melanocytes interact with keratinocytes by paracrine secretion of cytokines such as IL-1 / TNF-α, thereby promoting keratinocytes to express antimicrobial peptides (such as β-defensins). Therefore, the development of a cosmetic raw material that promotes melanogenesis by enhancing the innate immune response of melanocytes is of great significance in the treatment of hair graying.

[0004] A variety of plant extracts have been widely used in promoting the blackening of white hair. For example, patent CN104042752A discloses a composition for treating white hair, which is prepared from the following plant extracts by weight: 25-35 parts by weight of Polygonum multiflorum, 20-30 parts by weight of black sesame, 15-25 parts by weight of ginseng, 25-35 parts by weight of Cortex Moutan, 20-30 parts by weight of jujube, 20-30 parts by weight of Radix Rehmanniae, and 5-10 parts by weight of licorice. Patent CN109939061A discloses a safe and non-irritating essence for blackening white hair, which includes the following raw materials: water, flaxseed extract, angelica extract, aloe extract, tea polyphenols, ethanol, sodium benzoate, potassium sorbate, citric acid, ginseng extract, Polygonum multiflorum extract, γ-linolenic acid, glucomannan, and crude extract of cuttlefish ink. In addition, using fermentation technology to improve the efficacy of plant extracts has become a hot topic in the current cosmetics field. For example, the study on the effect of composite bacterial fermentation ginseng extract on the proliferation of mouse hair follicle cells [J] (Wang Jingxia et al., Central South Pharmacy, 2024, 22 (10), 2674-2679) found that the composite bacterial fermentation ginseng extract obtained after extraction with three bacterial species, Bacillus subtilis, Lactobacillus rhamnosus and Lactobacillus casei, had a proliferation rate of 59.93% on mouse hair follicle cells, which was better than the unfermented ginseng extract. Patent CN119033641A discloses a method for preparing plant fermentation products by multi-species combined fermentation, using Bacillus velez and Bacillus subtilis as a fermentation broth; extracting plant raw material tea bran by water extraction; inoculating and fermenting the water extract with a mixed bacterial broth of Bacillus velez and Bacillus subtilis; separating and purifying the fermentation broth to obtain the plant combined fermentation broth. This method can effectively increase the active substances in the fermentation broth and improve its antioxidant and other effects. However, none of the above-mentioned published patents or non-patent documents mention that the plant extract increases the production of melanin by enhancing the immune function of melanocytes.

[0005] In the production of fruit wine, yeast plays an important role and can be divided into two categories: brewing yeast and non-brewing yeast (such as abnormal Wickham yeast). Brewing yeast often dominates the early stage of fermentation due to its high ethanol production capacity and high tolerance. In contrast, abnormal Wickham yeast has the ability to produce a variety of flavor components and metabolites such as extracellular enzymes. It can more effectively decompose raw materials and convert these components into aroma-rich substances such as higher alcohols and esters. The synergistic interaction of these two yeasts can greatly improve the overall quality of fruit wine. However, Zhao Jianlei et al. found that in the mixed fermentation of abnormal Wickham yeast and brewing yeast, high concentrations of brewing yeast would cause abnormal Wickham yeast to stagnate and die in the early stage of fermentation through a cell-cell contact mechanism. Therefore, in mixed fermentation, the effect of the inoculation ratio of these two strains on the fermentation process and the product still needs further study.

[0006] So far, there is no research report on the use of a plant fermentation composition (Asparagus cochinchinensis, Polygonum multiflorum, black tea) prepared by co-fermentation of abnormal Wickham yeast and Saccharomyces cerevisiae in improving gray hair. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a plant fermentation composition for enhancing the scalp immune barrier and melanocyte activity, and its preparation and application. The invention uses microbial fermentation technology to provide a cosmetic raw material that promotes melanogenesis by enhancing the innate immune response of melanocytes, solving the technical problem of turning black hair white.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a plant fermentation composition for enhancing the scalp immune barrier and melanocyte activity, which is prepared by fermenting water extracts of Polygonum multiflorum, Asparagus cochinchinensis and black tea using abnormal Wickham yeast and Saccharomyces cerevisiae.

[0009] Asparagus, black tea and Polygonum multiflorum all have multiple pharmacological activities and biological effects. Asparagus polysaccharide activates immune response and innate immune memory through the TLR4-JNK / p38 / ERK signaling pathway, accompanied by the production of inflammatory factors, which may cause adverse skin reactions. Black tea can stimulate the proliferation of hair matrix keratinocytes by promoting the expression of insulin-like growth factor, prolong the hair growth cycle, and is beneficial to the production of hair follicle melanin in the hair growth period (stage III–VI). This may be due to the effect of caffeine contained in black tea, but different production processes affect the yield of caffeine. Polygonum multiflorum promotes the production of melanin in B16 cells by promoting the gene expression and protein synthesis of tyrosinase and microphthalmia-related transcription factors and activating the activity of tyrosinase. However, Polygonum multiflorum has certain toxicity, especially to the liver. For example, the 95% ethanol extract of Polygonum multiflorum can significantly inhibit the growth of human liver L-02 cells.

[0010] The fermentation method provided by the present invention utilizes the advantages of the metabolic activity of microorganisms and the enzyme catalytic system to enrich the effective ingredients in the raw materials, which can significantly improve the efficacy and safety of plant extracts. In addition to producing secondary metabolites due to fermentation to enhance the biological activity of plant extracts, microorganisms can also decompose and transform certain toxic components in plant extracts, converting toxic components into low-toxic or non-toxic substances, thereby reducing damage to the body. At the same time, microorganisms can secrete various extracellular enzymes to decompose the tight plant cell wall structure, enlarge the gaps between cells, and provide channels for the diffusion of substances inside and outside the cells, thereby improving the extraction rate and absorption utilization rate of active ingredients.

[0011] In some embodiments, the aqueous extract is prepared by: Step 1, drying Polygonum multiflorum, Asparagus cochinchinensis and black tea, and crushing and collecting powder; Step 2: Add water to the Polygonum multiflorum, Asparagus cochinchinensis and black tea powder obtained in step 1, perform ultrasonic-assisted extraction, separate the solid and liquid, and combine the filtrate to obtain the product.

[0012] Preferably, the weight ratio of Polygonum multiflorum, Asparagus cochinchinensis and black tea in step 1 is 10-30:1-10:1; more preferably 15-25:3-8:1.

[0013] Preferably, the drying in step 1 is: drying at 45°C-60°C for 3h-8h; more preferably, drying at 50°C-55°C for 4h-6h.

[0014] Preferably, the pulverization in step 1 is pulverizing until it passes through a 20-120 mesh sieve; more preferably, it passes through a 40-100 mesh sieve; and even more preferably, it passes through a 40-80 mesh sieve.

[0015] Preferably, the amount of water added in step 2 is 3-12 times the total amount of Polygonum multiflorum, Asparagus cochinchinensis and black tea powder; more preferably 5-10 times.

[0016] Preferably, the power of the ultrasonic-assisted extraction in step 2 is 200W-300W, the time is 20min-30min, and the number of extractions is at least 1 time; further preferably, the ultrasonic power is 250W-280W, and the time is 25min-30min.

[0017] In some more specific embodiments, the preparation method of the aqueous extract is: Step 1, respectively weigh 10-30 parts of Polygonum multiflorum, 1-10 parts of Asparagus cochinchinensis and 1 part of black tea, dry them in an oven at 50-55°C for 4h-6h, grind them through a 20-120 mesh sieve, and collect the powder; Step 2: Add water to the powders of Polygonum multiflorum, Asparagus cochinchinensis and black tea obtained in step 1 at a weight ratio of 1:3, perform ultrasonic-assisted extraction at 200W-300W for 20min-30min, separate the solid and liquid, and combine the filtrate.

[0018] In some embodiments, the ratio of the abnormal Wickham yeast to the Saccharomyces cerevisiae is 3-10:1, and more preferably 5-8:1.

[0019] In some more specific embodiments, the plant fermentation composition is prepared by fermenting water extracts of Polygonum multiflorum, Asparagus cochinchinensis and black tea at 26°C-32°C using abnormal Wickham yeast and Saccharomyces cerevisiae.

[0020] In a second aspect, the present invention provides a method for preparing the above-mentioned plant fermentation composition, comprising the following steps: S1. Substrate preparation: sterilize water extracts of Polygonum multiflorum, Asparagus cochinchinensis and black tea to obtain substrates; S2, fermentation: inoculating abnormal Wickham yeast and cerevisiae yeast into the substrate for fermentation to obtain a fermentation liquid; S3, separating the solid and liquid of the fermented liquid, filtering, and taking the filtrate.

[0021] In some embodiments, the inoculation amount of the abnormal Wickham yeast and the Saccharomyces cerevisiae in step S2 is 3%-10% of the weight of the substrate.

[0022] In some embodiments, the fermentation conditions in step S2 are: fermentation temperature 26°C-32°C, fermentation time 60h-120h.

[0023] In a third aspect, the present invention provides the use of the above-mentioned preparation method or the above-mentioned fermentation composition in the preparation of cosmetics.

[0024] In some embodiments, the cosmetic is a scalp care and / or hair care product.

[0025] In a fourth aspect, the present invention provides a cosmetic for improving the health of hair and scalp, comprising the above-mentioned plant fermentation composition.

[0026] The beneficial effects of the present invention are: (1) The plant fermentation composition of the present invention is prepared by fermenting the aqueous extract of Polygonum multiflorum, Asparagus cochinchinensis and black tea using abnormal Wickham's yeast and Saccharomyces cerevisiae, which is beneficial to the complementary synergistic system of the active components of Polygonum multiflorum, Asparagus cochinchinensis and black tea, and jointly exerts multiple functions such as inhibiting the growth of harmful bacteria, scavenging free radicals, enhancing the activity of melanocytes and melanin synthesis. At the same time, the synergistic effect of abnormal Wickham's yeast and Saccharomyces cerevisiae is utilized to convert the macromolecular active ingredients in the aqueous extract of Polygonum multiflorum, Asparagus cochinchinensis and black tea into small molecular active ingredients that are easily absorbed, thereby further improving the efficacy of the plant extract. (2) The plant fermentation composition of the present invention promotes the expression of β-defensins in keratinocytes and inhibits the production of inflammatory factor IL-6, thereby enhancing the scalp immune barrier.

[0027] (3) Cosmetics using the plant fermentation composition of the present invention can inhibit Propionibacterium acnes and Staphylococcus aureus, maintain the stability of the microbial community, regulate the microecological balance on the skin surface to enhance the scalp immune barrier, improve the health of hair and scalp, have a good hair darkening effect, and effectively prevent hair from turning gray.

[0028] (4) The plant fermentation composition of the present invention has antioxidant properties, which can reduce the damage caused by free radicals to hair follicle melanocytes. On the other hand, it can increase the expression of melanocyte Toll-like receptor 4, tyrosinase activity and promote melanin production, thereby achieving a better hair darkening effect from the two aspects of enhancing antioxidant capacity and promoting melanin production.

[0029] (5) The preparation method of the present invention is simple, safe, low-cost, and suitable for large-scale promotion and application. DETAILED DESCRIPTION

[0030] The following examples are only used to help understand the method of the present invention and its core idea. It should be noted that for those of ordinary skill in the art, without departing from the principles of the present invention, the present invention may also be subjected to several improvements and modifications, which also fall within the scope of protection of the claims of the present invention. The following description of the disclosed embodiments enables professionals and technicians in this field to implement or use the present invention. It will be apparent to professionals and technicians in this field that various modifications to these embodiments, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but may be applied to a wider range consistent with the principles and novel features disclosed herein. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the present invention belongs.

[0031] In addition to the above-mentioned basic ingredients, the optional components of the cosmetics of the present invention (plant fermentation composition) may also contain one or more known or other effective optional ingredients used in scalp care, hair care or personal care products, as long as these optional ingredients are compatible with the physical and chemical properties of the basic ingredients described herein, or do not excessively affect the stability, aesthetics or performance of the product. Non-limiting examples of such optional ingredients are described in the "Safety Technical Specifications for Cosmetics, 2015 Edition", which are incorporated herein by reference.

[0032] The purpose of the solid-liquid separation involved in the present invention is to achieve separation of the solid phase and the liquid phase. Therefore, conventional technical means such as centrifugation, filtration or filter pressing can be used.

[0033] Unless otherwise specified, all operations in this article were carried out at room temperature and the solvents used were water.

[0034] In the present invention, "melanin" and "melanochrome" have the same meaning and can be used interchangeably.

[0035] The following are examples and comparative examples of the present invention to explain the present invention in more detail, but the present invention is not limited thereto. The present invention does not limit the source of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all common commercial products in the technical field. The abnormal Wickerham yeast in the examples, Latin name Wickerhamomyces anomalus, was purchased from Guangdong Provincial Microbiological Culture Collection Center, numbered ZKCC12538, and the brewing yeast (Saccharomyces cerevisiae) was purchased from China Center for Type Culture Collection, with a collection number of CCTCC KY 2008613.

[0036] [Preparation of abnormal Wickham yeast solution and brewer's yeast solution] Streak abnormal Wickham yeast and Saccharomyces cerevisiae on YPD solid medium, incubate at 28±2℃ for 36h to restore the activity of the strains. Pick a single colony on the plate to 100 mL of sterilized YPD liquid medium, incubate at 28±2℃ and 150 rpm / min for 36 hours to obtain abnormal Wickham yeast seed liquid and Saccharomyces cerevisiae seed liquid, respectively. Take an appropriate amount of bacterial solution, dilute with sterile saline, and adjust the OD 600 value to the same range (OD 600 = 1.0) to ensure that the initial concentrations of the two yeasts were consistent.

[0037] Example 1 Preparation of plant fermentation composition S1. Take 10 parts of Polygonum multiflorum, 1 part of Asparagus cochinchinensis and 1 part of black tea by weight, dry them in an oven at 52±2℃ for 5 hours, grind them and pass through an 80-mesh sieve. Add distilled water to the powder at a weight ratio of 1:3, place it in an ultrasonic extractor, set the ultrasonic power to 200W and the time to 30min, centrifuge at 2500 rpm for 20 min after the ultrasonication, and obtain a water extract. Sterilize the water extract at 121℃ and high temperature and high pressure for 20min, and cool it to obtain the substrate.

[0038] S2. Mix 3 parts of abnormal Wickham yeast solution with 1 part of Saccharomyces cerevisiae solution in a weight ratio of 3:1 to prepare a mixed bacterial solution, inoculate the mixed bacterial solution into the substrate obtained in step S1 (the inoculation amount is 10% of the weight of the substrate), and ferment for 60 hours at a stirring speed of 150 rpm / min and a temperature of 30±2° C. to obtain a fermentation product.

[0039] S3. Centrifuge the fermentation product obtained in step S2 at a speed of 2500 rpm / min for 20 min, and filter it with a 0.22 μm ultrafiltration membrane to obtain a plant fermentation composition.

[0040] Example 2 Preparation of plant fermentation composition S1. Take 30 parts of Polygonum multiflorum, 10 parts of Asparagus cochinchinensis and 1 part of black tea by weight, dry them in an oven at 52±2℃ for 5 hours, grind them and pass through an 80-mesh sieve. Add distilled water to the powder at a weight ratio of 12:1, place it in an ultrasonic extractor, set the ultrasonic power to 300W and the time to 20min, centrifuge at 2500 rpm for 20min after the ultrasonication, and obtain a water extract. Sterilize the water extract at 121℃ and high temperature and high pressure for 30min, and cool it to obtain the substrate.

[0041] S2. In a weight ratio of 10:1, 10 parts of abnormal Wickham yeast solution and 1 part of cerevisiae yeast solution were mixed to prepare a mixed bacterial solution. The mixed bacterial solution was inoculated into the substrate obtained in step S1 (the inoculation amount was 3% of the weight of the substrate), and the mixture was fermented for 120 hours at a stirring speed of 150 rpm / min and a temperature of 28±2° C. to obtain a fermentation product.

[0042] S3. Centrifuge the fermentation product obtained in step S2 at a speed of 2500 rpm / min for 20 min, and filter it with a 0.22 μm ultrafiltration membrane to obtain a plant fermentation composition.

[0043] Example 3 Preparation of plant fermentation composition S1. Take 20 parts of Polygonum multiflorum, 5 parts of Asparagus cochinchinensis and 1 part of black tea by weight, dry them in an oven at 52±2℃ for 5 hours, and pass through an 80-mesh sieve after crushing. Add distilled water to the powder at a weight ratio of 8:1, place it in an ultrasonic extractor, set the ultrasonic power to 250W and the time to 25min, centrifuge at 2500 rpm for 20 min after the ultrasonication, and obtain a water extract. Sterilize the water extract at 121℃ and high temperature and high pressure for 20min, and cool it to obtain the substrate.

[0044] S2. Mix 6 parts of abnormal Wickham yeast solution with 1 part of Saccharomyces cerevisiae solution in a weight ratio of 6:1 to prepare a mixed bacterial solution, inoculate the mixed bacterial solution into the substrate obtained in step S1 (the inoculation amount is 5% of the weight of the substrate), and ferment for 90 hours at a stirring speed of 150 rpm / min and a temperature of 28±2° C. to obtain a fermentation product.

[0045] S3. Centrifuge the fermentation product obtained in step S2 at a speed of 2500 rpm / min for 20 min, and filter it with a 0.22 μm ultrafiltration membrane to obtain a plant fermentation composition.

[0046] Comparative Example 1 Preparation of plant fermentation composition The preparation steps and parameters of this comparative example are basically the same as those of Example 3, except that the yeast is only abnormal Wickham's yeast.

[0047] Comparative Example 2 Preparation of plant fermentation composition The preparation steps and parameters of this comparative example are basically the same as those of Example 3, except that the yeast is only Saccharomyces cerevisiae.

[0048] Comparative Example 3 Preparation of plant fermentation composition The preparation steps and parameters of this comparative example are basically the same as those of Example 3, except that the ratio of abnormal Wickham yeast and cerevisiae yeast in the mixed bacterial solution is different. Specifically, 2 parts of abnormal Wickham yeast solution and 1 part of brewer's yeast solution were mixed at a weight ratio of 2:1 to prepare a mixed bacterial solution.

[0049] Comparative Example 4 Preparation of plant fermentation composition The preparation steps and parameters of this comparative example are basically the same as those of Example 3, except that the ratio of abnormal Wickham yeast and cerevisiae yeast in the mixed bacterial solution is different. Specifically, 12 parts of abnormal Wickham yeast solution and 1 part of brewer's yeast solution were mixed at a weight ratio of 12:1 to prepare a mixed bacterial solution.

[0050] Comparative Example 5 Preparation of plant fermentation composition The preparation steps and parameters of this comparative example are basically the same as those of Example 3, except that the anomalous Wickham's yeast is replaced by Bifidosaccharomyces cerevisiae.

[0051] Comparative Example 6 Preparation of plant fermentation composition The preparation steps and parameters of this comparative example are basically the same as those of Example 3, except that the cerevisiae Saccharomyces cerevisiae in step (1) is replaced by bifid Saccharomyces cerevisiae.

[0052] Comparative Example 7 Preparation of plant fermentation composition The preparation steps and parameters of this comparative example are basically the same as those of Example 3, except that the weight ratios of Polygonum multiflorum, Asparagus cochinchinensis and black tea in the substrate are different; Specifically: weigh 35 parts of Polygonum multiflorum, 0.5 parts of Asparagus cochinchinensis and 1 part of black tea respectively.

[0053] Comparative Example 8 Preparation of plant fermentation composition The preparation steps and parameters of this comparative example are basically the same as those of Example 3, except that the weight ratios of Polygonum multiflorum, Asparagus cochinchinensis and black tea in the substrate are different.

[0054] Specifically: weigh 8 parts of Polygonum multiflorum, 12 parts of Asparagus cochinchinensis and 1 part of black tea respectively.

[0055] Comparative Example 9 Preparation of plant composition According to weight, 20 parts of Polygonum multiflorum, 5 parts of Asparagus cochinchinensis and 1 part of black tea were taken and dried in an oven at 52±2℃ for 5 hours, crushed and passed through an 80-mesh sieve. Distilled water was added to make the material-liquid ratio 5-10:1, placed in an ultrasonic extractor, and the ultrasonic power was set to 250W and the time was 25min. After the ultrasonication, centrifuged at 2500 rpm for 20 min to obtain a water extract, and the water extract was sterilized at 121℃ and high temperature and high pressure for 20 min, and cooled to obtain.

[0056] Effect Example 1: Enhance scalp immunity 1. Viability detection of keratinocytes (HaCaT) In this experiment, a blank group (DMEM culture medium), a cell control group (DMEM culture medium + HaCaT cells) and a sample group (DMEM culture medium + HaCaT cells + test samples provided in Examples 1 to 3 and Comparative Examples 1 to 9) were set up for cell viability testing, and each group had 3 parallel wells.

[0057] HaCaT cells in logarithmic growth phase were cultured at 1×10 5 The cells were seeded at a density of 100 / mL in a 96-well plate. 2 Remove the culture medium, add 100 μL of sample solution to each well, and continue to incubate at 37°C and 5% CO 2 Incubate for 24 hours under the conditions of . Add 20 μL of MTT solution to each well and continue incubation for 2 hours. Discard the supernatant and add 100 μL of isopropanol to each well. Oscillate at 100 r / min for 30 minutes at room temperature in the dark. Use an ELISA reader to detect the absorbance of each well at a wavelength of 570 nm.

[0058] Calculate cell viability according to the following formula, screen the sample mass concentration with cell viability greater than 90% and no morphological changes, and conduct subsequent efficacy evaluation research.

[0059]

[0060] Among them, OD(sample group) represents the absorbance of the sample group, OD(blank group) represents the absorbance of the blank group, and OD(cell control group) represents the absorbance of the cell control group.

[0061] The results are shown in Table 1.

[0062] Table 1

[0063] The results showed that when the concentration was 5 mg / mL, the cell viability of each group of samples was maintained above 90%, indicating that the plant fermentation composition of the present invention had no obvious cytotoxic effect and could be used for the determination of IL-6 and HBD-2 content.

[0064] 2. Determination of interleukin-6 (IL-6) and human beta-defensin-2 (HBD-2) content IL-6 content determination test grouping: set up a control group (DMEM), a model group (1 μg / mL LPS + cells + DMEM), a positive control group (1 μg / mL LPS + 35 μM quercetin + cells + DMEM), and a sample group (1 μg / mL LPS + 5 mg / mL test samples provided in Examples 1-3 and Comparative Examples 1-9 + cells + DMEM) to determine the IL-6 content, with 3 parallel wells in each group.

[0065] HBD-2 content determination test grouping: set up a control group (DMEM), a sample group (5 mg / mL test sample provided by Example 1-3 and Comparative Example 1-9 + HaCaT cells + DMEM), and a positive control group (100 mg / mL L-arginine) to determine the HBD-2 content, and each group has 3 parallel wells.

[0066] HaCaT cells in logarithmic growth phase were cultured at 1×10 4 The cells were seeded at a density of 10 cells / mL in a 96-well plate. 2 Remove the culture medium and add 500 μL of sample solution diluted with DMEM medium to each well. Continue to incubate at 37°C and 5% CO 2 Incubate for 24 hours under the conditions of . The supernatant was centrifuged for 3 minutes (4°C, 12000 r / min).

[0067] According to the operating instructions of the interleukin-6 (IL-6) and human β-defensin-2 (HBD-2) kits, after adding the sample, incubate and wash the plate, add the enzyme reagent, incubate and wash the plate again, perform the color reaction, and terminate the reaction. Detect the IL-6 and HBD-2 content in the supernatant on the machine: draw the standard curve, and calculate the concentration of IL-6 and HBD-2 in the cell culture supernatant of each group (in pg / mL).

[0068] The inhibition rate of IL-6 and the promotion rate of HBD-2 were calculated according to the following formulas. The higher the inhibition rate of IL-6, the stronger the ability of the plant fermentation composition to inhibit the inflammatory response of HaCaT cells; the higher the promotion rate of HBD-2, the stronger the ability of the plant fermentation composition to enhance the innate immune response of HaCaT cells, which can enhance the immune function of the scalp.

[0069]

[0070] The results are shown in Table 2.

[0071] Table 2

[0072] The results showed that the plant fermentation composition provided by the present invention can enhance the scalp immune function by enhancing the expression of β-defensin-2 (HBD-2); at the same time, it can inhibit the secretion of IL-6 in HaCaT cells to avoid excessive inflammatory response, and has the effect of enhancing the scalp immune barrier.

[0073] Effect Example 2: Regulating the microecological balance on the scalp surface Evaluation of antibacterial activity: The antibacterial effect of the plant fermentation composition on Propionibacterium acnes (ATCC11827) and Staphylococcus aureus (ATCC6538) was analyzed using the Oxford cup punch method.

[0074] Take the above strains and make them into 2×10 5 CFU / mL bacterial suspension. The bacterial suspension and culture medium are added to the plate and mixed thoroughly. A sterile Oxford cup is placed on the surface of the culture medium and a hole is punched. After solidification, 100 μg of the sample to be tested (5 mg / mL test sample provided by Example 1-Example 3 and Comparative Example 1-Comparative Example 9) is added. Three parallel wells are set in each group. Staphylococcus aureus is cultured under aerobic conditions at 37±2°C for 2 days, and Propionibacterium acnes is cultured under anaerobic conditions at 37±2°C for 2 days. After culture, the diameter of the inhibition zone is measured with a vernier caliper, and the antibacterial effect is evaluated by the diameter of the inhibition zone. If the diameter of the inhibition zone of the sample to be tested is >8 mm, it has an antibacterial effect; if the diameter of the inhibition zone of the sample to be tested is ≤8 mm, it has no antibacterial effect.

[0075] The results are shown in Table 3.

[0076] Table 3

[0077] The results show that the plant fermentation compositions prepared in Examples 1 to 3 of the present invention can simultaneously inhibit Propionibacterium acnes and Staphylococcus aureus, maintain the stability of the microbiome, and have the effect of regulating the microecological balance of the skin surface.

[0078] By comparing the comparative examples 1 to 9 and the example 3, it can be seen that the plant fermentation compositions prepared in the comparative examples 1 to 9 cannot effectively inhibit Propionibacterium acnes and Staphylococcus aureus, indicating that the combined fermentation of abnormal strains of Wickham yeast and brewer's yeast and the amount of plant ingredients (Asparagus cochinchinensis, Polygonum multiflorum and black tea) during the fermentation process have a great influence on the antibacterial effect of the obtained plant fermentation composition. Effect Example 3: Enhancement of the activity of melanoma B16 cells Melanoma B16 cells were diluted with culture medium to a concentration of 0.8 × 10 cells per well. 4 The cells were plated in a 96-well plate at a density of 100 μL. After culturing for 24 h, 100 μL of sample solution was added to the blank control group (DMEM medium), cell control group (melanoma B16 cells + DMEM medium) and sample group (melanoma B16 cells + test samples provided in Examples 1-3 and Comparative Examples 1-9 + DMEM medium) in turn. The culture was continued for 24 h. 10 μL of CCK-8 solution was added to each well and incubated at 37 °C in CO 2 The cells were incubated in an incubator for 3 h, and the absorbance (OD) at 450 nm was measured using a microplate reader.

[0079] The cell viability (%) of melanoma B16 cells treated with the test samples provided in Examples 1 to 3 and Comparative Examples 1 to 9 was calculated according to the following formula.

[0080]

[0081] Among them, OD(sample group) represents the absorbance of the test sample group, OD(blank control group) represents the absorbance of the blank control group, and OD(cell control group) represents the absorbance of the cell control group.

[0082] The results are shown in Table 4.

[0083] Table 4

[0084] The results show that the samples provided by the plant fermentation composition in Example 1-Example 3 have a cell viability greater than 100% in the concentration range of 1.25-5 mg / mL, which can promote the proliferation of mouse melanoma B16 cells. And as the concentration of the test sample increases, the viability of B16 cells gradually increases. However, the test samples provided by Comparative Examples 1-Example 9 failed to enhance the viability of B16 cells. Among them, the test samples provided by Examples 1-Example 3 and Comparative Examples 1-Example 9 have the maximum viability of B16 cells at a treatment concentration of 5 mg / mL, which is greater than 90%, and no morphological changes are observed, and can be used to carry out subsequent efficacy evaluation studies.

[0085] Effect Example 4: Enhancement of the antioxidant capacity of melanoma B16 cells In this experiment, a blank control group (melanoma B16 cells + DMEM medium), a model group (melanoma B16 cells + DMEM medium), a positive control group (melanoma B16 cells + 5 mg / mL vitamin C + DMEM medium) and a sample group (melanoma B16 cells + test samples provided in Examples 1-3 and Comparative Examples 1-9 + DMEM medium) were set up for ROS testing, with 6 parallel wells in each group. Melanoma B16 cells were diluted with DMEM medium, with 100 μL of cell suspension per well (the number of cells per well was 1×10 4 ) were dripped into a 96-well plate, and when the cell density reached 70% to 80%, the sample group and the model group were treated with 150 μmol / L H 2 O 2 After 10 min of treatment, the experimental group was added with the test sample, and the blank control group and model group were added with serum-free DMEM medium. 2Incubate in the incubator for 30 min, then dilute DCFH-DA (2',7'-dichlorofluorescein diacetate fluorescent probe) with serum-free medium to a final concentration of 5 μmol / L, incubate in a 37 ℃ incubator for 30 min, wash the cells three times with serum-free cell culture medium to fully remove DCFH-DA that has not entered the cells, and use a microplate reader to detect the absorbance at an excitation light of 488 nm and an emission light of 530 nm.

[0086] According to the following formula, the cell viability (%) of melanoma B16 cells treated with the test samples provided in Examples 1 to 3 and Comparative Examples 1 to 9 was calculated. Compared with the model group, a high ROS inhibition rate indicates a strong antioxidant capacity, which is more conducive to reducing the damage caused by oxidative stress to melanocytes.

[0087]

[0088] The results are shown in Table 5.

[0089] Table 5

[0090] The results show that the plant fermentation compositions provided in Examples 1 to 3 of the present invention can effectively reduce ROS levels, have strong antioxidant capacity, and can effectively reduce the oxidative damage to hair follicle melanocytes caused by free radicals, prevent the occurrence of gray hair and other problems, and the effect is significantly better than that of positive drugs.

[0091] By comparing the comparative examples 1 to 2 and the example 3 group, it can be seen that in the present invention, only abnormal Wickham yeast or saccharomyces cerevisiae is used to ferment the water extract of Polygonum multiflorum, Asparagus cochinchinensis and black tea, and the ROS inhibition rate of the prepared plant fermentation composition is only 58.7%-66.2%. In the example 3 group, the water extract of Polygonum multiflorum, Asparagus cochinchinensis and black tea is fermented by a combination of abnormal Wickham yeast and saccharomyces cerevisiae, and the ROS inhibition rate of the prepared plant fermentation composition reaches 94.8%, indicating that the combination of abnormal Wickham yeast and saccharomyces cerevisiae in the present invention produces a synergistic effect in antioxidant capacity.

[0092] Comparison of Comparative Examples 3 to 6 and Example 3 shows that in the present invention, the ROS inhibition rate of the plant fermentation composition prepared by fermenting the aqueous extract of Polygonum multiflorum, Asparagus cochinchinensis and black tea with a conventional amount of abnormal Wickham yeast and Saccharomyces cerevisiae or a conventional yeast is only 60.9%-70.8%, which is much lower than that of Example 3. This indicates that the plant fermentation composition prepared by the conventional amount of abnormal Wickham yeast and Saccharomyces cerevisiae or a conventional yeast cannot produce a synergistic effect in terms of antioxidant capacity.

[0093] By comparing Comparative Examples 7-8 and Example 3, it can be seen that in the present invention, the dosage of Polygonum multiflorum, Asparagus cochinchinensis and black tea has a greater impact on the antioxidant capacity of the plant fermentation composition, and the water extract prepared with conventional dosages of Polygonum multiflorum, Asparagus cochinchinensis and black tea cannot achieve the technical effect of the invention.

[0094] Effect Example 5: Promoting the expression of Toll-like receptor 4 (TLR4) in melanoma B16 cells GAPDH (internal reference) and TLR4 upstream and downstream sequences were purchased from Beijing Qingke Biotechnology Co., Ltd. In this experiment, the blank control group (DMEM culture medium) and the sample group (5 mg / mL plant fermentation composition provided by Example 1-Example 3 and Comparative Example 1-Comparative Example 9 + DMEM culture medium) were used to treat mouse melanoma B16 cells for 48 hours. RNA was extracted from the above samples using an RNA extraction kit. NANO-400A ultra-micro nucleic acid analyzer was used to detect RNA concentration and purity. According to the instructions of the reverse transcription kit, the reverse transcription reaction system was prepared on ice. After reverse transcription of RNA into cDNA on a PCR instrument according to the instructions on the reverse transcription kit, the real-time fluorescence quantitative PCR (RT-PCR) reaction system was prepared on ice: 5 μL of RT-PCR SYBR Green, 0.2 μL of upstream and downstream primers, 1 μL of sample cDNA, and 3.6 μL of double distilled water were taken on a 96-well plate dedicated to fluorescence quantitative PCR; the RT-PCR reaction conditions of Bio-rad were: 95 ℃ pre-denaturation for 2 min, 95 ℃ denaturation for 10 s, 60 ℃ annealing / extension for 30 s, and 40 cycles. According to the obtained Ct value, 2-△△Ct calculation analysis was used. After three repeated experiments, the activation rate of TLR4 was calculated according to the following formula.

[0095]

[0096] The higher the activation rate, the stronger the ability of the plant fermentation composition to enhance the response of melanocytes to innate immune stimulation.

[0097] The results are shown in Table 6.

[0098] Table 6

[0099] The results show that the plant fermentation compositions provided in Examples 1 to 3 of the present invention have All of them have activation effects, which are significantly better than Comparative Examples 1 to 9. This indicates that the plant fermentation composition provided by the present invention can promote the expression of Toll-like receptor 4 (TLR4) in melanoma B16 cells, and promote melanogenesis by participating in the innate immune response.

[0100] Effect Example 6: Enhanced tyrosinase activity Cell culture and experimental treatment are the same as in Example 4. Set up enzyme kinetics experimental groups: control group (DMEM medium + B16 cells) and sample group (DMEM medium + B16 cells + test samples provided in Example 1-Example 3 and Comparative Example 1-Comparative Example 9), use 200 μL reaction system, set 5 replicate wells for each concentration, and culture for 48 h. Discard the supernatant and wash twice with PBS. Add 80 μL of PBS buffer containing 1% TritonX-100 to each well and quickly place it in a -80℃ low-temperature refrigerator for 1 h. Take it out and place it to melt at room temperature, incubate it at 37℃ for 5 min, add 20 μL of 0.5% L-dopa by mass fraction, react at 37℃ for 6h, and measure the absorbance value at 490 nm on an enzyme marker.

[0101] The enzyme activity was calculated according to the following formula: tyrosinase activity = OD value of experimental group / OD value of control group × 100%.

[0102]

[0103] The higher the activation rate of tyrosinase, the stronger the ability of the plant fermentation composition to promote melanin production and the better the hair darkening effect.

[0104] The results are shown in Table 7.

[0105] Table 7

[0106] The results show that the plant fermentation compositions provided in Examples 1 to 3 all have the effect of activating tyrosinase, and are significantly better than Comparative Examples 1 to 9.

[0107] At the same time, by comparing the comparative examples 1 to 2 and the example 3 group, it can be seen that in the present invention, only abnormal Wickham yeast or saccharomyces cerevisiae is used to ferment the water extract of Polygonum multiflorum, Asparagus cochinchinensis and black tea, and the prepared plant fermentation composition has a weak effect on activating tyrosinase, with an activation rate of only 35.2%-37.8%. In the example 3 group, abnormal Wickham yeast and saccharomyces cerevisiae are used to ferment the water extract of Polygonum multiflorum, Asparagus cochinchinensis and black tea, and the activation rate of the prepared plant fermentation composition for tyrosinase reaches 89.6%, indicating that the abnormal Wickham yeast and saccharomyces cerevisiae in the present invention The combination of abnormal Wickham yeast and saccharomyces cerevisiae produces a synergistic effect in activating tyrosinase.

[0108] Comparison of Comparative Examples 3 to 6 and Example 3 shows that in the present invention, the activation rate of tyrosinase in the plant fermentation composition prepared by fermenting the aqueous extract of Polygonum multiflorum, Asparagus cochinchinensis and black tea with a conventional amount of abnormal Wickham yeast and Saccharomyces cerevisiae or a conventional yeast is no more than 40%, which is much lower than that of Example 3. This indicates that the conventional amount of abnormal Wickham yeast and Saccharomyces cerevisiae or the conventional yeast cannot produce a synergistic effect in activating tyrosinase.

[0109] Effect Example 7: Increase the melanin content of melanocytes Cell culture and experimental grouping were the same as in Example 4. 100 μL of B16 melanoma cell suspension was added to a 96-well plate to a cell density of 0.5×10 4 Each well was plated with 5 replicate wells and cultured in an incubator (37°C, 5% carbon dioxide) for 12 h. Then, 1 mL of culture medium without the test sample was added to the blank control group and the negative control group, and 1 mL of culture medium containing the corresponding test sample (concentration 5 mg / mL) was added to the sample group. Each group had 5 replicate wells and was cultured in an incubator (37°C, 5% carbon dioxide) for 56 h.

[0110] After the culture was completed, the culture medium was removed, the cells were digested with 0.25% trypsin, and centrifuged at 1000r / min for 5 minutes. The supernatant was discarded to obtain the precipitate. 200μL of 1mol / LNaOH solution containing 10% (DMSO) was added to the B16 cells of each treatment group, and the cells were shaken for 5 minutes to completely dissolve the intracellular melanin particles. The absorbance value at 490nm was measured using a spectrophotometer to calculate the melanin content. Each group was measured 3 times and the average value was taken.

[0111]

[0112] Among them, OD1 is the absorbance value of the sample group, OD2 is the absorbance value of the negative control group, and OD3 is the absorbance value of the blank control group.

[0113] The results are shown in Table 8.

[0114] Table 8

[0115] The results showed that compared with the negative control group, the melanin content in the B16 melanoma cells of Example 1-Example 3 groups was significantly increased (P<0.05), indicating that the plant fermentation composition prepared in Example 1-Example 3 has the effect of promoting melanin production.

[0116] Compared with the negative control group, the melanin content in the B16 melanoma cells of the comparative examples 1 to 9 groups did not change significantly (P>0.05), indicating that the plant fermentation compositions prepared in the comparative examples 1 to 9 did not have the effect of promoting melanin production.

[0117] The above is a further description of the present invention in conjunction with specific embodiments, but these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

Claims

1. A method for preparing a plant fermentation composition for enhancing scalp immune barrier and melanocyte activity, characterized in that: The method comprises the following steps: using water extracts of Polygonum multiflorum, Asparagus cochinchinensis and black tea as substrates, and fermenting with abnormal Wickham yeast and Saccharomyces cerevisiae; The ratio of abnormal Wickham yeast to Saccharomyces cerevisiae is 3-10:1; The preparation method of the water extract is: Step 1, drying Polygonum multiflorum, Asparagus cochinchinensis and black tea, and crushing and collecting powder; Step 2: Add water to the Polygonum multiflorum, Asparagus cochinchinensis and black tea powder obtained in step 1, perform ultrasonic-assisted extraction, separate the solid and liquid, and combine the filtrate; wherein the weight ratio of the Polygonum multiflorum, Asparagus cochinchinensis and black tea is 10-30:1-10:

1.

2. The preparation method according to claim 1, characterized in that: The mass ratio of the abnormal Wickham yeast to the brewer's yeast is 5-8:1; the weight ratio of the Polygonum multiflorum, Asparagus cochinchinensis and black tea is 15-25:3-8:

1.

3. The preparation method according to claim 1, characterized in that: The inoculation amount of the abnormal Wickham yeast and the brewer's yeast is 3%-10% of the weight of the substrate.

4. The preparation method according to claim 1, characterized in that: The amount of water added in step 2 is 3 to 12 times the total amount of Polygonum multiflorum, Asparagus cochinchinensis and black tea powder. The power of ultrasonic assisted extraction in step 2 is 200W to 300W, and the time is 20min to 30min.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The steps include: S1. Substrate preparation: sterilize the water extracts of Polygonum multiflorum, Asparagus cochinchinensis and black tea to obtain substrates; S2, fermentation: inoculating abnormal Wickham yeast and cerevisiae yeast into the substrate for fermentation to obtain a fermentation liquid; S3, separating the solid and liquid of the fermented liquid, filtering, and taking the filtrate.

6. The preparation method according to claim 5, characterized in that: The fermentation conditions in step S2 are: fermentation temperature 26°C-32°C, fermentation time 60h-120h.

7. A plant fermentation composition prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the preparation method according to any one of claims 1 to 6 or the plant fermentation composition according to claim 7 in the preparation of cosmetics.

9. The use according to claim 8, characterized in that: The cosmetic is at least one of a scalp care product and a hair care product.

10. A cosmetic for improving the health of hair and scalp, characterized in that: The invention comprises the plant fermentation composition according to claim 7.

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